This project aims to assess the importance of changes in intracellular free calcium concentration, [Ca++]i, in the response to light of intact vertebrate rod photoreceptors. We hope to determine (1) what is [Ca++]i in rod outer segments (ROS) in darkness and light; 2) if [Ca++]i does change on illumination, as current theories predict, does it correlate with stimulus intensity and with the conductance change in ROS plasma membranes; 3) to what extent are [Ca++]i changes driven by plasma membrane fluxes or by internal stores; 4) can [Ca2+]i be altered by means other than light, and if so, how are rod properties affected. The novel methodology is based on a new family of calcium chelators with most of the ideal properties of [Ca++]i indicator and a method for trapping controlled quantities of these chelators, up to several millimolar, in the cytoplasm of a whole population of intact cells (Tsien, 1980, 1981). Preliminary studies indicate that this method, based on intracellular hydrolysis of membrane-permeable esters, does work in principle in rods, but needs some chemical optimization of the ester groups. Also the chelators need further development to make operating wavelengths as long as possible. Toad or salamander rods would then be loaded with the [Ca++] indicator, whose absorbance or preferably fluorescence would be monitored along with membrane potential or outer segment current of one representative rod. We would hope to correlate [Ca++]i with input variables such as stimulus light intensity and duration, extracellular ion concentrations (especially of CA++ and Na+ , pharmacological agents including ionophores for CA++ and modifiers of cyclic nucleotide metabolism, and indicator content varying from non-buffering levels to high levels which should damp down any [Ca++]i transients. [Ca++]i would also be compared with simultaneously measured electrical responses. Such experiments could help greatly in defining what [Ca++]i does in rods and in deciding between models for phototransduction in vertebrate retina.